Long non‐coding RNA MALAT1 regulates retinal neurodegeneration through CREB signaling

This partial retraction replaces the editorial note from February 2022. Journal editors became aware of potential image aberrations in the figures in August 2021. At the time, the editors had notified the authors’ institution to request an investigation into the aberrations. The investigation concluded that image processing errors affecting Figs 3B and 7C and D, Appendix Figs S2 and S5 resulted in images being mislabeled and misrepresented. Readers were alerted in February 2022 that the authors were repeating experiments to address aberrations in the figures. The committee determined that the conclusions derived from the repeat experiments were consistent with the conclusions presented in the original paper. The data

Authors' response to Journal.

th September 2021
Dear editor: We just received the following e-mail about our image aberrations in EMM-2015-05725-V4. You mentioned the following " We have carefully checked your figures and the source data you provided at publication against one another and have come across several inconsistencies that need to be addressed". Could you tell us which images have aberrations? This paper has experienced more than 4 times revision. At the beginning, the number of biolgoical repicates was less than 5. During the revision, the reviewers and editor asked us to proivde more biological repicates. We conducted more experiments to provide more replicates. This revision may lead to some inconsistencies. We are very for these errors. If required, we can provide the original data and the relevant repicates. As for the inconsistencies, we can reconduct the relevant experiment and provide new results.
Finally, we can assure you that we have carefully conduct this experiment and do not have academic misconducts. We are so sorry for the relevant unintentional errors. We hope you can give a chance to correct these errors.
Notebly, the key findings of this paper has been verfied in the following studies published in recent years, such as Targeting long non-coding RNA MALAT1 alleviates retinal neurodegeneration in diabetic mice (Int J Ophthalmol. 2020; 13 (2) figures. The affected figures are 3B, 7C, 7D, S2 and S5. Please review these figures yourself and send us the related source data. Our goal is to ensure that the scientific record is accurate, and we are interested in working with you and your institute to do so. We have recently become aware of potential image aberrations in their paper, and we conducted a standard image analysis in which we found additional inconsistencies in the figures. The detected issues concern staining image reuse within and between figures. The authors sent us source data, but we have no way to validate it.
In cases of serious image aberrations that could undermine the conclusions of the paper, we notify the authors' institution to provide an opportunity for quality control and investigation at the institutional level. We have a policy to cooperate with institutions whenever possible, and in our experience, this can help identify the causes of the apparent aberrations since institutions can directly view lab books and interview the authors.
I have attached here a summary of our image analysis results and the source data provided by the authors with my comments. I would be happy to discuss these issues in a further conversation, either via e-mail, video call or telephone. I kindly ask that you update us within the next two weeks to inform us about further steps from your side. I'm looking forward to hearing from you.
Please note that we have informed Dr. Yan in parallel that we have contacted you on this matter. We welcome your response at your earliest convenience.

Institute to Journal 11 th October 2021
Thanks for giving us an opportunity for quality control and investigation at the institutional level. As the head of the academic committee of Eye Hospital, I and my colleagues have viewed the lab books and interviewed the first and corresponding authors. We also carefully view all source data. The survey reports are shown below: (1) For PKC and Rhodopsin staining in Fig S2 and  (2) For background process problems: WB background ( These replicates have similar expression pattern. These 5 different replicates were conducted at different time points and at different instruments. Thankfully, they only adjusted the background of dots, but did not process the dot bands. The final results were expressed as the relative change of band gray value compared with control group. This statistical method was not affected by the background of WB. Thus, the final conclusion was not affected.
For Fig 5G untreated group, this problem is associated with the processing of IF images from gray model to RGB model. The authors also provided the relevant source data. Generally, there was no apoptotic cells in untreated group. Thus, this inappropriate processing would not affect the final conclusion. The authors have provided the source data for these images. There were a lot of original images for these experiments. During the selection of representative images, they mistakenly selected the representative images from the same group, which led to the wrong choices of representative images. In addition, they took more than one photos for a slice. They did not move the slice enough between the intervals of photo capture, which led to the duplications between different photos.
For 7C and D, the authors have provided the enough replicates and the relevant source data. For Fig 3B and S7B, they have also provided other replicates. In addition, the results of nestin and vimentin in Figure 3C, 3D, and S8 could provide additional evidence to support their findings in Fig 3B and S7B. Given the final conclusion was determined by the bar graphs but not the representative images, the wrong selected representative images did not affect the final conclusion.
The third-party verification: To further investigate whether the conclusion is believable, the authors have provided the third-party evidences to verify the main findings in this paper. 2) PACAP attenuates optic nerve crush-induced retinal ganglion cell apoptosis via activation of the CREB-Bcl-2 pathway (Journal of Molecular Neuroscience, 2019 They also reported that CREB in retinal ganglion cell apoptosis) Taken together, the authors could provide the source data for these relevant images and there were no data integrity issue. However, due to their careless and paying insufficient attention to supplementary materials, they have made the relevant errors.
Subsequently, we suggest that the journal can allow the authors to publish an erratum to correct these errors. If required, they could use the stored samples to re-conduct the relevant experiments to verify the conclusion. I and our colleagues could supervise the process of re-conducted experiments.

Journal to authors 2 nd November 2021
Thank you for your patience. We have now had the chance to re-discuss your paper and would like to request you to repeat the experiments under the supervision of the investigative committee, as suggested by Dr. Shang. After the experiments are done we can decide how to proceed based on the results. How long would it take you to repeat the experiments with the original samples?

Authors to Journal 4 th November 2021
Thank you for giving us the chance to repeat the experiments under the supervision by Dr. Shang.
Today, we had a meeting and discussed with the graduate students who will be responsible for the subsequent experiments. They tell us that they will take about 3 months to complete all experiments. The time required for the relevant experiments as shown below: • Western blots: 2-3 weeks for the relevant blots with five replicates; • PI staining: 2-3 weeks including cell thawing and PI staining with 4 replicates; • IF staining and HE staining for tissue slices: about 3 weeks; • Electron microscope experiments: about 4-5 weeks. We required advance reservation for electron microscope in another institute (about 4-week advance reservation).
Today, they also carefully checked the original samples. Fortunately, we have all relevant protein samples and embedded tissue samples. We are afraid of some antibodies may not work because these antibodies were purchased in 2014. If they could not work, we will buy the same antibody from the same company with the same catalogues number. Due to the antibody is usually not in stock in China and require imports, it may take more time due to the effects of Covid-19.
Taken together, we will try our best to complete all experiments within three months. If more questions, please feel free to contact us. In line with the journal policy, we had notified the research institution for quality control at the institutional level and requested the authors to repeat the experiments under the supervision of the investigative committee. Meanwhile, we have issued an editorial note attached to the paper to alert readers to these image aberrations and an ongoing effort from the authors to repeat the experiments (https://www.embopress.org/doi/full/10.15252/emmm.202115623).

Authors to
The authors have now repeated the experiments, provided new figures and analyses. We have conducted a forensic image analysis on the repeat data and did not detect aberrations in these new images. Since the errors occur in figures that are essential to the main conclusion of the paper(specifically Figure 7), I would be grateful if you could take a look at the new figures and analyses and let us know whether you think that they seem acceptable or if, in your opinion, the main conclusions and findings are compromised.
In particular, I would like to point you to several issues we noticed in the new data, and we would appreciate your input on them: 1. In their previous paper (https://www.embopress.org/doi/full/10.15252/emmm.201505725), the authors used Mann-Whitney U-test to calculate the p-value. In their repeat experiments, they performed One-way ANOVA tests. In your view, is the statistical method switch acceptable?
2. In the legend of Figure S2: the authors provided p-values for ONT+scr vs. WT and ONT+M vs. WT, instead of comparing ONT+scr and ONT+M to ONT. Similarly, in Figure S5, 2. In the legend of Figure S2: the authors provided p-values for ONT+scr vs. WT and ONT+M vs. WT, instead of comparing ONT+scr and ONT+M to ONT. Similarly, in Figure S5, they compared DR+ scr or DR+M with WT. Do you think such comparison and analysis are appropriate?
Since the fluorescence intensity is not different between WT or ONT, it won't make a difference. I think showing the change from WT is good, but they should include the p-values for all groups compared/tested, which will include the original comparison. They will have all of that information already if they used ANOVA. Based on the graph values, it shouldn't change any significance shown in their new figure.
3. In the Rhodopsis experiments in the original Appendix Figures S2 and S5: WT shows a significant difference from the other tested samples. However, WT is not significantly different from the other experiments in the repeat experiments. Is this discrepancy concerning to you, or do you think the authors can clarify this?
This one is interesting, and I apologize that I didn't catch it years ago when I reviewed the paper. Based on their results/conclusions in the manuscript, the WT should not have been different. Unless optic nerve transection causes a loss of the photoreceptor cells, which I'm not aware of from my experience in the field. I believe the new data provided is more accurate, but the authors should clarify the discrepancy in those results and why they initially had a significant decrease from WT.
Additional Comments: ONT is mislabeled in the graph for Supplementary figure 2 with the PKCa staining.
For figure 7D, can they make sure to include how they counted the abnormal axons? Was it just by eye from an investigator for the 3 images they took per group? Or was software used to detect the abnormalities? Did they add up the abnormalities from all three images or average them? I'm guessing add, since they have a max of approximately 40 for the new data and almost 90 for the published figure (but used 10 images instead of three for the analysis). They should make sure to state how they counted and whether the investigator was blinded or aware of the groupings. We recently became aware of potential image aberrations in this paper, so we asked the authors to repeat some of the experiments. The authors now repeated the experiments and provided new figures and analyses. However, in their previous article, the authors used Mann-Whitney U-test, and in their repeat experiments, they performed One-way ANOVA tests. We asked one of the reviewers who reviewed the original paper to comment on this, and here is their response: "They are looking at multiple groups, so Mann-Whitney was not the correct test to be used. Thank you very much in advance. I would be very grateful if you could provide us with your expert opinion on this matter.

Statistics expert to Journal 3 rd May 2022
One-way ANOVA between two groups is the t-test. Looking at the plots, this looks fine. Also, it should not matterqualitatively similar results should also come out with the Mann-Whitney U-test. But for several theoretical and conceptual reasons, the t-test (or ANOVA test) is actually preferable.
I think the statement "They are looking at multiple groups, so Mann-Whitney was not the correct test to be used." is not pertinent, since even although there are >2 groups, the comparisons are always pairwise.
Perhaps, to make sure your readers do not get the same (unfounded) doubts as your reviewer, the authors could be asked to provide the p-values for that, too. The basic results should be consistent.

Journal's response to authors 7 th June 2022
Thank you for sending us your repeat data and analysis. We have now gone through your data and response in detail. As you will see below, we have also invited one of the referees who had evaluated the original manuscript to review the repeat data. In particular, we asked them specifically to comment on several issues that we noticed.
I have included below our questions and the referee's response. Considering these comments, we would ask you to address the following issues: • Point #2: please include the p-values for all groups compared/tested. • Point #3: please clarify the discrepancy in those results.
• Address the additional comments from the referee about Figure 7.
• Please note that for point #1, we also consulted with a statistician who confirmed that the current one-way ANOVA test is appropriate.
Please send us a revised version of the repeat data and a point-by-point response to the referee's comments. Feel free to let me know if you have any questions.

Author's response 20 th June 2022
Thank you for sending us your repeat data and analysis. We have now gone through your data and response in detail. As you will see below, we have also invited one of the referees who had evaluated the original manuscript to review the repeat data. In particular, we asked them specifically to comment on several issues that we noticed.
I have included below our questions and the referee's response. Considering these comments, we would ask you to address the following issues: -Point #2: please include the p-values for all groups compared/tested.

Response:
We all included the p-values for all groups compared/tested for figure S2 and figure S5.  -Point #3: please clarify the discrepancy in those results.

Response:
We first explained why Rhodopsin expression in WT group was altered in the repeated experiment.
Rhodopsin is a G-protein coupled receptor found in the rod cells In fact, the visual chromophore 11-cis-retinal binds to opsin to form rhodopsin in the dark, which locks the opsin in an inactive state. Unbound free opsin is known to be constitutively In the repeated experiment, the experiment was conducted by a different student. The previous student has been graduated and leaved. We also have moved our lab and the animal culture condition was also changed. Thus, these external factors may affect the expression of Rhodopsin in WT group. As for Rhodopsin experiments in Figures S2 and S5, the purpose of these experiments was to investigate whether MALAT1 knockdown affected the photoreceptors in diabetic retinas or ONT mice. The expression of Rhodopsin between ONT+M group and ONT group or between ONT+M group and ONT+Scr group did not show significant difference. The expression of Rhodopsin between DR+M group and DR group or between DR+M group and DR+Scr group did not show significant difference. Based on the above-mentioned results, we could conclude that the final conclusion was not associated with WT group. And, the current result did not alter the total conclusion of this manuscript.
-Address the additional comments from the referee about Figure 7.

Response:
Three ultra-thin cross sections per nerve were observed and added together to count the number of damaged axons. Counting of damaged axons was performed by three different investigators blinded to group identity and injury status. An average counting number of the three investigators was used for statistical analysis.
-Please note that for point #1, we also consulted with a statistician who confirmed that the current one-way ANOVA test is appropriate.

Response:
According to the suggestion of the statistician, the t-test (or ANOVA test) is actually preferable, we used the One-way ANOVA followed by Dunnet's multiple comparison test for statistical analysis.
Additional comments from the reviewer "ONT is mislabeled in the graph for Supplementary figure 2 with the PKCa staining.

Response:
We have corrected it. "NOT" has been changed to "ONT" For figure 7D, can they make sure to include how they counted the abnormal axons?
Was it just by eye from an investigator for the 3 images they took per group? Or was software used to detect the abnormalities? Did they add up the abnormalities from all three images or average them? I'm guessing add, since they have a max of approximately 40 for the new data and almost 90 for the published figure (but used 10 images instead of three for the analysis). They should make sure to state how they counted and whether the investigator was blinded or aware of the groupings.

Response:
Three ultra-thin cross sections per nerve were observed and added together to count the number of damaged axons. Counting of damaged axons was performed by 3 different investigators blinded to group identity and injury status. An average counting number of the 3 investigators was used for statistical analysis.

Journal's response 15 th July 2022
Thank you for sending us the most recent point-by-point. We are satisfied with the repeat data and plan to move forward to correct your paper.
We would selectively retract Figures 3B, 7C-D, Appendix Figures S2 and S5, and publish the repeat data in the retraction notice. We cannot replace the data in the original manuscript because the repeat experiments were not part of the original experimentation.
We've drafted a partial retraction note (attached) and have included space for you to make an author statement. We would ask you to return the document with your modifications by 29th of July. After we have agreed on a final draft, we will send the notice to all authors and request that everyone signals whether they agree or disagree with the notice and the partial retraction.
In addition, we would require the figure legends included in the word document, and production-quality figures for the repeat data.
Thank you for your collaboration to come to a resolution on this case.

th July 2022
Dear editor: Thanks for giving us the chance for correction.
We have provided the relevant files in the attachment, including (1) a partial retraction note (2) Figure legend (3) production-quality figures for the repeat data for Figures 3B, 7C